Both growth factors (stimulators of cell cycling) and growth inhibitory factors (inhibitory factors of cell cycling) play an important role in cell-cell interactions and cell division. Despite the practical importance of growth inhibitory factors, very few have been isolated and purified and, in most cases, there is little evidence that the biologically active inhibitory factors previously isolated are residents of the cell surface or act by binding cell surface receptors. The existence of cell surface inhibitory factors would be consistent with the models of regulation of cell division as a result of cell-cell contact.
Compared to the numerous growth factors that have been described, very few inhibitory factors of cell proliferation have been isolated and characterized. The major inhibitory factors described include: 1) the 25 kDa homodimer transforming growth factor-.beta. (TGF-.beta.) that has mitogenic activity with a variety of fibroblasts and yet expresses a potent inhibitory activity with normal human epithelial prokeratinocytes cultured in serum-free medium (Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82: 119-123, 1985; Coffey et al., Cancer Res. 48: 1596-1602, 1988) and the structurally-related protein isolated from African green monkey cells (BCS-1) conditioned medium (Tucker et al., Science 226: 705-707, 1984); 2) a 12-14 kDa protein isolated from mammary tissue (Bohmer et al., Exp. Cell Res. 150; 466-476, 1984; Muller et al., J. Cell. Physiol. 138: 415-423, 1989) that has been identified in cell nuclei and shown to be structurally related to a fibroblast growth inhibitory factor isolated from mouse 3T3 cell medium (Voss et al., Exp. Cell Res. 138: 397-407, 1982; Bohmer et al, J. Cell. Biochem. 38: 199-204, 1988); 3) a 17 kDa acidic protein, originally described as a glial maturation factor .beta., that has been shown to have antiproliferative activity (Lim, Proc. Natl. Acad. Sci. U.S.A. 86: 3901-3905, 1989; Lim et al., Cell Regulat. 1: 741-746, 1990); 4) an oligosaccharide from human diploid fibroblasts (Wieser et al., J. Cell Biol. 111: 2681-2692, 1990); 5) a tissue-specific growth inhibitory factor (mammostatin) isolated from cell culture medium (Ervin et al., Science 244: 1585-1587, 1989); and, 6) two classes of glycopeptide inhibitory factors structurally unrelated to the present invention (Kinders et al., Exp. Cell Res. 136: 31-41, 1981; Charp et al., J. Cell Biol. 97: 311-316, 1983).
Cell proliferation inhibitory factors that are membrane residents and that play a role in cell-cell signaling, most likely have significant hydrophobic domains, or are complexed with hydrophobic integral membrane components. This feature has led to technical difficulties in their isolation, identification, and their presentation to target cells for meaningful biological assays--particularly when detergents (necessary to maintain the elements in aqueous suspension or solution) are toxic solvents to living cells.
A hydrophilic and active fragment of a larger glycoprotein inhibitory factor was released from intact cells that allowed purification by biochemical procedures (Sharifi et al., J. Chromat. 324: 173-180, 1985; Sharifi et al., Neurochem. 46: 461-469, 1986a).
The bovine inhibitory glycopeptide is composed of a single polypeptide chain of a molecular weight of approximately 18,000 that focuses by isoelectric focusing at about 3.0 (Sharifi et al, Neurochem. 46: 461-469, 1986; and Sharifi et al, J. Cell. Biochem. 31: 41-47, 1986). The glycopeptide inhibits cellular protein and DNA synthesis, and arrests cells in the mitotic cycle at what appears to be a single block point near the G.sub.1 /S interphase (Fattaey et al., J. Cell. Physiol. 139: 269-274, 1989; and Fattaey et al, Exper. Cell Res. 194: 62-68, 1991). The glycopeptide inhibitory factor requires only a cell surface interaction to mediate its biological inhibitory activity (Sharifi et al., Biochem. Biophys. Res. Comm. 134: 1350-1357, 1986c), and the binding kinetics are consistent with a specific and saturable cell surface receptor (Bascom et al., J. Cell Physiol. 128: 202-208, 1986; Sharifi and Johnson, J. Biol. Chem. 262: 15752-15755, 1987).
Consistent with the hypothesis that cell division is controlled by the interaction of ligands at the cell surface with both positive and negative influences, the glycopeptide has been identified on the surfaces of 3T3 cells (Lakshmanarao et al., Exper. Cell Res. 195: 412-415, 1991), and to be a potent antagonist of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (Chou et al., Cancer Lett. 35: 119-128, 1987), epidermal growth factor (EGF) (Bascom et al., J. Cell. Biochem. 34: 283-291, 1987) and bombesin (Johnson and Sharifi, Biochem. Biophys. Res. Comm. 161: 468-474, 1989).
Although the glycopeptide was isolated from bovine cerebral cortex cells, its inhibitory action is effective on wide range of target cells. Cells sensitive to its proliferative inhibitory action include vertebrate and invertebrate (insect) cells, fibroblast and epithelial-like cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines (Fattaey et al., J. Cell. Physiol. 139: 269-274, 1989; and Fattaey et al, Exper. Cell Res. 194: 62-68, 1991).
With the exception of one cell line, human HL-60 leukemic cells, all cells which were inhibited were reversibly inhibited by the glycopeptide in a nontoxic manner (Edson et al, Life Sci. 48: 1813-1820, 1991). HL-60 cells, however, were arrested in an irreversible fashion although they remained viable for at least 84 h. The glycopeptide mediated a terminal cellular differentiation, even after its removal.
An interesting feature of the glycopeptide is that the biological inhibitory activity clearly is Ca.sup.2+ dependent, and possibly related to cellular Ca.sup.2+ fluxes and/or intracellular Ca.sup.2+ mobilization (Toole-Simms et al., J. Cell. Physiol. 147: 292-297, 1991). The addition of the calcium ionophore A23187, but not the sodium ionophore monensin, before or within minutes of the inhibitory factor, results in the abrogation of the inhibition of protein synthesis (Sharifi et al., Biochem. Biophys. Res. Comm. 136: 976-982, 1986).
Prior to the subject invention, a particularly disturbing feature of the purified glycopeptide was that a protease activity, of unknown specificity, always was measurable in even the most purified preparations (Sharifi et al., J. Cell. Biochem. 31: 41-47, 1986). Whether the protease was an integral activity of the glycopeptide molecule itself, or a trace contaminant in the purified preparations could not be determined. Although the protease activity remained even when the biological inhibitory activity of the glycopeptide was destroyed (Sobieski et al., Life Sci. 38: 1883-1888, 1986), the proteases presence was unavoidable and complicated the preparation of samples for studies of protein sequencing.